Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 129 4 ACKNOWLEDGEMENTS We thank Dr. Mike Boxem for providing the human cDNA library; Rian Stoffelen for her contributions to Fig 1E and F; and Ginny C. Farias Galdames and Arthur P.H. de Jong for critically reading the article. This work was supported by the Netherlands Organization for Scientific Research (NWO-ALW-VICI, CCH; NWO-VIDI, MA), the Netherlands Organization for Health Research and Development (ZonMW-TOP, CCH), the European Research Council (ERC; ERC-consolidator, CCH; ERC-StG, HDM), and the Proteins@Work program of the National Roadmap Large-scale Research Facilities of the Netherlands (MA). AUTHOR CONTRIBUTIONS FWL designed, conducted, and interpreted experiments, and wrote the article. YC performed biochemical experiments and cloned constructs. JTK initiated the study, cloned constructs, and performed initial experiments together with MMB. AB conducted the electric field stimulation experiments, RS performed the mass spectrometry experiments and was supported by MA, and EAK provided the expansion microscopy data. HDM gave advice throughout the project and edited the article. CCH supervised the research, coordinated the study, and edited the article. CONFLICT OF INTEREST CCH is an employee of Genentech, Inc., a member of the Roche group. The authors declare that they have no additional conflict of interest. MATERIALAND METHODS Animals AllanimalexperimentswereapprovedbytheAnimalEthicalReviewCommittee(DEC)ofUtrecht University and performed in accordance with the guidelines for the welfare of experimental animals issuedby theDutch lawand followingEuropean regulations (Guideline86/609/EEC). Primary rat neuron culture and transfection Dissociated hippocampal and cortical neuron cultures were prepared from embryonic day 18 rat pups of mixed gender. Cells were plated on 18-mm glass coverslips coated with poly-l-lysine (37.5 mg/ml) and laminin (1.25 mg/ml) in a 12-well plate at a density of 100k/ well for hippocampal neurons and 50k/well for cortical neurons. Cultures were maintained in Neurobasal medium (NB) supplemented with 2% B27, 0.5 mM glutamine, 16.6 μM glutamate, and 1% penicillin/streptomycin at 37°C in 5% CO2. Neuron cultures were transfected using a mixture of 3.3 μl lipofectamine 2000 (Invitrogen), 1.5–3.0 μg DNA, and 200 μl NB per coverslip. For knockdown experiments, a total of 1.5 μg DNA of shRNA construct(s) was used per coverslip. The transfection mixture was added to the coverslips, which were placed in fresh NB supplemented with 0.5 mM glutamine, and incubated for 45–90 min. Next, coverslips were washed once in prewarmed NB and